Comparative analysis of small extracellular vesicles in peritoneal fluid from endometriosis and non-endometriosis...

Aim: Endometriosis is a chronic inflammatory disorder requiring clinical examination, imaging, and surgical intervention for accurate assessment. The aim of the present study was to provide a detailed biophysical and biochemical characterization of small extracellular vesicles (sEVs) derived from peritoneal fluid, and to explore disease-associated molecular differences that may inform future investigations. Methods: sEVs were isolated from the peritoneal fluid of women with endometriosis (n = 7), women without endometriosis who underwent surgery for benign, noninflammatory conditions (n = 7), and follicular fluid samples (n = 3). The tetraspanin profile of the samples was determined using single-particle interferometric reflectance imaging sensing. After size exclusion chromatography and ultracentrifugation, atomic force microscopy (AFM) and Raman spectroscopy were employed to characterize the sEVs. Results: Prominent Raman spectral variations in peritoneal sEVs between control women and those with endometriosis were observed at 891 cm^-1 (proteins) and 968 cm^-1 (lipids, CH wagging). Statistical analysis of molecular components influencing the Raman signature of sEVs suggest that their lipid composition appears to be influenced by the pathophysiology of endometriosis, reflecting changes similar to those observed in the eutopic endometrium in previous studies. sEVs isolated from peritoneal fluid and follicular fluid have markedly distinct biochemical compositions and morphometric properties. sEVs from follicular fluid were significantly smaller in size and exhibited a higher rigidity in this pilot cohort. Conclusion: Distinct biophysical and biochemical differences in sEVs from endometriosis patients are documented in this pilot study, contributing to a deeper characterization of disease-associated extracellular vesicle (EV) alterations. Variations between follicular and peritoneal fluid sEVs suggest unique functional roles in reproductive health. These findings are hypothesis-generating and warrant validation in larger, well-controlled cohorts before any diagnostic application can be claimed.